The eukaryotic pterin-dependent hydroxylases each catalyze physiologically critical reactions. Phenylanine hydroxylase catalyzes the hydroxylation of phenylalanine to tyrosine during catabolism. Mutations in phenylalanine hydroxylase result in phenylketonuria. Tyrosine hydroxylase catalyzes the formation of dihydroxyphenylalanine from tyrosine, the first step in biosynthesis of the catecholamine neurotransmitters. Imbalances in catecholamine levels have been implicated in a number of disease states, including hypertension. Tryptophan hydroxylase catalyze the hydroxylation of tryptophan, the first step in the biosynthesis of serotonin. Imbalances in serotonin levels have been implicated in several neurological and psychiatric disorders, including depression. Thus, these enzymes have central roles in the health of an individual. Despite their importance, little is known about them at a molecular level. The long term goals of the research proposed here are to determine the catalytic mechanisms of the pterin dependent hydroxylases and to determine at a molecular level the effects of phosphorylation on tyrosine hydroxylase. The specific aims of the present proposal are to examine the mechanisms using alternative substrates, probe the role of the active site iron in oxygen activation, determine the role of active site residues using site-directed mutagenesis and the recently determined structures, and further characterize the effects of phosphorylation on tyrosine hydroxylase.